Genome-Wide Characteristics of GH9B Family Members in Melon and Their Expression Profiles under Exogenous Hormone and Far-Red Light Treatment during the Grafting Healing Process

β-1,4-glucanase can not only promote the wound healing of grafted seedlings but can also have a positive effect on a plant’s cell wall construction. As a critical gene of β-1,4-glucanase, GH9B is involved in cell wall remodeling and intercellular adhesion and plays a vital role in grafting healing. However, the GH9B family members have not yet been characterized for melons. In this study, 18 CmGH9Bs were identified from the melon genome, and these CmGH9Bs were located on 15 chromosomes. Our phylogenetic analysis of these CmGH9B genes and GH9B genes from other species divided them into three clusters. The gene structure and conserved functional domains of CmGH9Bs in different populations differed significantly. However, CmGH9Bs responded to cis elements such as low temperature, exogenous hormones, drought, and injury induction. The expression profiles of CmGH9Bs were different. During the graft healing process of the melon scion grafted onto the squash rootstock, both exogenous naphthyl acetic acid (NAA) and far-red light treatment significantly induced the upregulated expression of CmGH9B14 related to the graft healing process. The results provided a technical possibility for managing the graft healing of melon grafted onto squash by regulating CmGH9B14 expression.


Introduction
Grafting technology has been used as a conventional method in breeding fruit trees and vegetable seedlings [1,2], which can significantly improve a single variety's yield, quality, and tolerance. It has been widely used in the cultivation and variety improvement of horticultural crops and created enormous economic value [3]. Due to the significance of grafted seedlings, there is a heightened curiosity in the grafting and recovery of grafted seedlings with a high survival rate. Melon (Cucumis melo L.) is an important economic fruit crop grown worldwide, with an annual output of more than 27 million tons in 2019 [4]. It originated in Africa and India [5] and is considered a rich source of multiple nutrients, such as vitamins, minerals, and dietary fiber [6]. Presently, melon grafting cultivation is a successful strategy for managing soil-borne diseases, overcoming the issue of continuous cropping, and boosting yield. Cucurbita moschata (C. moschata) is a genus of Cucurbitaceae squash that originated from Mexico to Central America, and it is widely cultivated around the world [7]. Grafting improved the absorption and utilization of water and nutrients due to the more developed root system [8], and grafted plants had resistance to a variety of Int. J. Mol. Sci. 2023, 24, 8258 2 of 17 soil-borne diseases. Therefore, squash is often used as the rootstock for grafting. However, in the actual production, due to the poor affinity between scion and rootstock, and long healing management cycle, the grafted seedlings have a low commercial rate and high seedling raising cost, which seriously affect the cultivation and production costs of grafted melon seedlings [9].
It was shown that the overexpression of β-1,4-glucanase promoted grafting [10], and a branch secreted into the extracellular region promoted cell wall reconstruction near the grafting interface. The GH9 in plants has three branches (GH9A, GH9B, and GH9C) [11][12][13]. In avocado and pepper, the GH9B gene caused the fruit to stop growing and soften the tissue, which is related to the changes in the cell wall structure. In tomatoes, silencing of SlGH9B2 increased the force required to separate the fruit from the plant [14]. In Arabidopsis, the Cel3 and Cel5 genes of GH9Bs were thought to promote cell wall loosening required for root cap cell shedding during root development and respond to biotic and abiotic rhizosphere stress. In poplar, the overexpression of GH9Bs reduced the amount of xylan cross-linked with cellulose microfibers and increased cell wall plasticity. The cellulose's crystallinity and polymerization degrees were decreased by the overexpression of the OsGH9B1 and OsGH9B3 in rice, but the digestibility of cell wall enzymes was increased [15]. In poplar and Arabidopsis, the GH9B5 gene was highly expressed during the development of the secondary wall, enhancing the cell wall structure [16]. During grafting between tobacco and Arabidopsis families, a large number of genes encoding cell wall modification enzymes were induced by transcription. Among them, the expression of the GH9B3 gene encoding β-1,4-glucanase subclass increased continuously and promoted cell wall adhesion [17]. In tobacco, NbGH9B3 was induced, along with other wall-remodeling genes at these graft junctions. The dysregulation of NbGH9B3 significantly reduced the success rate of multiple homografts and heterografts [18]. In Solanaceae, the regulation mechanism of GH9B3 gene expression has been changed. The expression of GH9B genes coding β-1,4-glucanase was relatively conserved in the grafting process of soybean and morning glories. The GH9B3 gene improved the graft compatibility of petunias with different families and then enhanced the reproduction technology and flower yield [19]. During the interaction of parasitic plants, S. japonicum secreted the direct homolog of the PjGH9B3 gene around the haustrum and directly contacted the host tissue, which reduced the thickness of the host wall at the interface between parasite and host plant cells and formed a xylem bridge to promote successful cell adhesion [20]. GH9B genes were demonstrated to be vital in the healing of grafted plants. The GH9B genes isolated and identified in Arabidopsis were divided into ten families (GH9B1 family, GH9B3 family, GH9B7 family, GH9B8 family, GH9B10 family, GH9B11 family, GH9B12 family, GH9B15 family, GH9B16 family, and GH9B18 family). Based on the phylogenetic classification of the GH9B family in Arabidopsis, the evolutionary relationships of the GH9B family members in other plant species can be analyzed using the same method. As plant genomic data continue to be published, members of the GH9B family will be identified from more and more plant species, which will lay the foundation for studying these genes from an evolutionary biological perspective.
The GH9B gene is essential for promoting graft healing and plant growth. However, the melon's GH9B family members have not been thoroughly examined and characterized at the genomic level. We identified GH9B family members from melon genomes to improve the healing of the melon grafted onto the squash. This study aimed to investigate the structure and function of the GH9B gene in melon and the phylogenetic relationship between the melon protein and Arabidopsis thaliana. To determine the CmGH9Bs expression pattern, we deeply evaluated their responses to exogenous hormones and far-red light signaling. This study provided a theoretical basis for the functional analysis of GH9B family genes in melon that was of great importance for further regulating the healing of the melon grafted onto the squash.

Identification of Members of the CmGH9B Gene Family Members
We identified 18 CmGH9Bs from the genome of melon genes and all GH9B genes mapped onto the melon chromosomes. According to the chromosome gene distribution order, they were named from CmGH9B01 to CmGH9B18. The characteristics of CmGH9Bs, including the molecular weight, PI, instability index, and subcellular location of the 18 CmGH9Bs, were summarized in Table 1. The number of amino acids composing these CmGH9Bs varied greatly. The CmGH9B1 comprised only 172 amino acids with a molecular mass range of 19,277.59 Da, and the CmGH9B12 consisted of 722 amino acids with a molecular mass range of 79,876.41 Da. The CmGH9B16 had the lowest PI (4.95), while the CmGH9B11 had the highest PI (9.41). There were only nine genes, namely CmGH9B1, CmGH9B4, CmGH9B6, CmGH9B7, CmGH9B8, CmGH9B12, CmGH9B16, CmGH9B17, CmGH9B18, in the cell membrane; only two genes, namely CmGH9B9 and CmGH9B14, in the cell wall; and seven genes, namely CmGH9B2, CmGH9B3, CmGH9B5, CmGH9B10, CmGH9B11, CmGH9B13, and CmGH9B15, in both the cell membrane and the cell wall (Table 1).

Phylogenetic Analysis, Classification, and Conserved Domain Analysis of CmGH9Bs
To study the phylogenetic relationships of 18 CmGH9Bs, we constructed phylogenetic trees based on amino acid sequence identity ( Figure 1). The 18 CmGH9Bs were divided into three subtribes (I, II, and III), of which the largest group was Subtribe III (15 members), and Subtribes I and II contained one and two members, respectively ( Figure 1A). Subtribe I included CmGH9B1, Subtribe II had CmGH9B7 and CmGH9B18, and the rest belonged to Subtribe III. The conserved domain analysis revealed that all CmGH9Bs contained the Clyco-Hydro-9 domain. However, not all CmGH9Bs had the CBM49 domain; it was mainly in Subtribe III ( Figure 1B). Moreover, we represented the phylogenetic tree of relationships to analyze the evolutionary relationship between 18 CmGH9Bs and 10 AtGH9Bs. The results showed that these GH9B genes were divided into three subtribes (I-III). Subgroup III was the largest group, with 15 CmGH9Bs and 10 AtGH9Bs, the same subtribe and classification as CmGH9B alone ( Figure 1C). Therefore, we speculated that CmGH9Bs and AtGH9Bs shared a common evolutionary origin, and CmGH9B14 was the homologous gene of AtGH9B3. subtribe and classification as CmGH9B alone ( Figure 1C). Therefore, we speculated that CmGH9Bs and AtGH9Bs shared a common evolutionary origin, and CmGH9B14 was the homologous gene of AtGH9B3.

Motif and Exon-Intron Structure Analysis of CmGH9Bs
To determine the function of CmGH9Bs, the motif compositions of 18 CmGH9Bs were analyzed by the amino acid sequence in the MEME program. An analysis of 18 CmGH9Bs ( Figure 2A) showed that, apart from CmGH9B18 (no Motif2), CmGH9B14 (no Motif3 or Motif5), and CmGH9B1 (Subtribe I; no Motif1, -2, -3, -4, -5, -8, and -10), all the other motifs were present in the other 15 CmGH9Bs. These determine the classification, structure, and function of CmGH9Bs. Although their role has not been elucidated, they may indicate that the GH9B gene in melon has multiple functions. To identify the differences between the GH9B gene family in melon, we analyzed the CmGH9Bs' structure by comparing each coding sequence with the corresponding genome sequence. As shown in Figure 2, the numbers of CmGH9Bs exons were discontinuously and unevenly distributed from 1 to 18. Combining the gene structure and phylogenetic tree, we found that the exon-intron distribution of CmGH9Bs was related to their classification. Closely related genes usually had homologs. Therefore, their genetic structure was similar. For example, the CmGH9B15 in the second subfamily had no introns, only exons. However, the CmGH9Bs, except for CmGH9B15, had exons and introns. Subgroup I had fewer exons (4 to 7) than other subgroups, and the exon-intron structural characteristics of Subgroups II and III were similar ( Figure 2B). These results suggested that although CmGH9Bs were subdivided into three families, they were relatively genetically conserved.

Motif and Exon-Intron Structure Analysis of CmGH9Bs
To determine the function of CmGH9Bs, the motif compositions of 18 CmGH9Bs were analyzed by the amino acid sequence in the MEME program. An analysis of 18 CmGH9Bs ( Figure 2A) showed that, apart from CmGH9B18 (no Motif2), CmGH9B14 (no Motif3 or Motif5), and CmGH9B1 (Subtribe I; no Motif1, -2, -3, -4, -5, -8, and -10), all the other motifs were present in the other 15 CmGH9Bs. These determine the classification, structure, and function of CmGH9Bs. Although their role has not been elucidated, they may indicate that the GH9B gene in melon has multiple functions. To identify the differences between the GH9B gene family in melon, we analyzed the CmGH9Bs' structure by comparing each coding sequence with the corresponding genome sequence. As shown in Figure 2, the numbers of CmGH9Bs exons were discontinuously and unevenly distributed from 1 to 18. Combining the gene structure and phylogenetic tree, we found that the exon-intron distribution of CmGH9Bs was related to their classification. Closely related genes usually had homologs. Therefore, their genetic structure was similar. For example, the CmGH9B15 in the second subfamily had no introns, only exons. However, the CmGH9Bs, except for CmGH9B15, had exons and introns. Subgroup I had fewer exons (4 to 7) than other subgroups, and the exon-intron structural characteristics of Subgroups II and III were similar ( Figure 2B). These results suggested that although CmGH9Bs were subdivided into three families, they were relatively genetically conserved.

Distribution of CmGH9Bs in Chromosomes
According to the melon genomic data, chromosome mapping of CmGH9Bs was carried out. The extraction of the chromosomal information of the CmGH9Bs identified their chromosomal locations. As shown in Figure 3, all CmGH9Bs had precise positions in the chromosomes. Each melon chromosome contains ≥1 CmGH9B. The CmGH9Bs were unevenly and non-randomly distributed on 15 chromosomes. Chr3 (chromosomal 3) had the four CmGH9Bs, and the rest of the Chr included only one CmGH9B. Chr3 was the longest in melon and consisted of many CmGH9Bs. The shortest chromosome, Chr14, had one CmGH9B. There was no significant correlation between chromosome length and CmGH9B gene distribution. There was no apparent correlation between the chromosome length and CmGH9Bs' distribution ( Figure 3). exon-intron of CmGH9Bs. Round green rectangles, black lines, and yellow rectangles, respectively, marked the exons, introns, and untranslated regions. The scale bar at the bottom estimated the lengths of the exons, introns, and untranslated regions.

Distribution of CmGH9Bs in Chromosomes
According to the melon genomic data, chromosome mapping of CmGH9Bs was carried out. The extraction of the chromosomal information of the CmGH9Bs identified their chromosomal locations. As shown in Figure 3, all CmGH9Bs had precise positions in the chromosomes. Each melon chromosome contains ≥1 CmGH9B. The CmGH9Bs were unevenly and non-randomly distributed on 15 chromosomes. Chr3 (chromosomal 3) had the four CmGH9Bs, and the rest of the Chr included only one CmGH9B. Chr3 was the longest in melon and consisted of many CmGH9Bs. The shortest chromosome, Chr14, had one CmGH9B. There was no significant correlation between chromosome length and CmGH9B gene distribution. There was no apparent correlation between the chromosome length and CmGH9Bs' distribution ( Figure 3).

Cis-Acting Element Analysis of CmGH9Bs Promoters
To gain insight into the genetic functions, metabolic networks, and regulatory mechanisms of melon trihelix genes' potential function of CmGH9Bs, the hypothetical promoters of cis-acting elements in 18 CmGH9Bs promoters (upstream 2 KB) were predicted and identified. The 18 CmGH9Bs had 627 cis-acting elements that reacted to the various abiotic stresses. Moreover, these cis-acting elements responded to gibberellin (GA), salicylic acid (SA), abscisic acid (ABA), methyl jasmonate (ME-JA), and so on. (Figure 4). Photoresponsive elements were detected in 18 melon mate promoters, accounting for the most significant proportion of all melon mate promoters. Among the 627 cis-acting factors, 19 cisacting factors were associated with ME-JA response, distributed to 13 CmGH9Bs. The eight cis-acting factors were related to auxin response and distributed to six CmGH9Bs. The nine cis-acting factors were associated with GA response and distributed to seven CmGH9Bs. The seven cis-acting factors were related to SA response, allocated to six CmGH9Bs. Based on the above analysis, it was speculated that the CmGH9Bs played essential roles in response to the hormones.

Cis-Acting Element Analysis of CmGH9Bs Promoters
To gain insight into the genetic functions, metabolic networks, and regulatory mechanisms of melon trihelix genes' potential function of CmGH9Bs, the hypothetical promoters of cis-acting elements in 18 CmGH9Bs promoters (upstream 2 KB) were predicted and identified. The 18 CmGH9Bs had 627 cis-acting elements that reacted to the various abiotic stresses. Moreover, these cis-acting elements responded to gibberellin (GA), salicylic acid (SA), abscisic acid (ABA), methyl jasmonate (ME-JA), and so on. (Figure 4). Photoresponsive elements were detected in 18 melon mate promoters, accounting for the most significant proportion of all melon mate promoters. Among the 627 cis-acting factors, 19 cis-acting factors were associated with ME-JA response, distributed to 13 CmGH9Bs. The eight cis-acting factors were related to auxin response and distributed to six CmGH9Bs. The nine cis-acting factors were associated with GA response and distributed to seven CmGH9Bs. The seven cis-acting factors were related to SA response, allocated to six CmGH9Bs. Based on the above analysis, it was speculated that the CmGH9Bs played essential roles in response to the hormones.

Expression Patterns of the 18 CmGH9Bs in the Scion Stem during the Graft Healing Process
To better understand the expression pattern of 18 CmGH9Bs during graft healing, we measured and analyzed their relative expression in the melon scion stem of melon grafted onto squash rootstock by using qRT-PCR technology ( Figure 5). The results showed that the relative expression level of CmGH9B18, belonging to Subtribe II, was the highest and significantly higher than the other 17 CmGH9Bs. In Subtribe III, the relative expression of CmGH9B12 was markedly higher than the other 14 CmGH9Bs; the relative expression levels of CmGH9B4, CmGH9B5, CmGH9B8, CmGH9B13, CmGH9B15, and CmGH9B17 were low, and there was no significant difference between them.

Expression Patterns of the 18 CmGH9Bs in the Scion Stem during the Graft Healing Process
To better understand the expression pattern of 18 CmGH9Bs during graft healing, we measured and analyzed their relative expression in the melon scion stem of melon grafted onto squash rootstock by using qRT-PCR technology ( Figure 5). The results showed that the relative expression level of CmGH9B18, belonging to Subtribe II, was the highest and significantly higher than the other 17 CmGH9Bs. In Subtribe III, the relative expression of CmGH9B12 was markedly higher than the other 14 CmGH9Bs; the relative expression levels of CmGH9B4, CmGH9B5, CmGH9B8, CmGH9B13, CmGH9B15, and CmGH9B17 were low, and there was no significant difference between them.

Expression Patterns of the 18 CmGH9Bs in the Scion Stem during the Graft Healing Process
To better understand the expression pattern of 18 CmGH9Bs during graft healing, we measured and analyzed their relative expression in the melon scion stem of melon grafted onto squash rootstock by using qRT-PCR technology ( Figure 5). The results showed that the relative expression level of CmGH9B18, belonging to Subtribe II, was the highest and significantly higher than the other 17 CmGH9Bs. In Subtribe III, the relative expression of CmGH9B12 was markedly higher than the other 14 CmGH9Bs; the relative expression levels of CmGH9B4, CmGH9B5, CmGH9B8, CmGH9B13, CmGH9B15, and CmGH9B17 were low, and there was no significant difference between them.

Effects of Exogenous Hormones on the Expression Pattern of 18 CmGH9Bs
Based on the cis-acting element analysis of 18 CmGH9Bs promoters, it was speculated that the CmGH9Bs played essential roles in response to the hormones. Thus, we analyzed the effects of exogenous NAA treatment on 18 CmGH9Bs expression in the scion stem of the melon grafted onto the squash rootstock ( Figure 6). The results indicated the relative expression levels of six CmGH9Bs, namely CmGH9B2, CmGH9B6, CmGH9B7, CmGH9B14, CmGH9B16, and CmGH9B17, that were significantly upregulated; and the relative expression levels of two CmGH9Bs, namely CmGH9B4 and CmGH9B8, that were significantly downregulated. There were no significant differences for the other 10 CmGH9Bs (CmGH9B1, CmGH9B3, CmGH9B5, CmGH9B9, CmGH9B10, CmGH9B11, CmGH9B12, CmGH9B13, CmGH9B15, and CmGH9B18) under exogenous NAA treatment. Notably, the relative expression level of CmGH9B14, a function analogous to AtGH9B3 ( Figure 1C), was significantly increased with the exogenous NAA application. Nevertheless, further research and evidence are necessary to ascertain whether NAA treatment can enhance the graft compatibility of melon scion and squash rootstock and promote its healing.

Effects of Exogenous Hormones on the Expression Pattern of 18 CmGH9Bs
Based on the cis-acting element analysis of 18 CmGH9Bs promoters, it was speculated that the CmGH9Bs played essential roles in response to the hormones. Thus, we analyzed the effects of exogenous NAA treatment on 18 CmGH9Bs expression in the scion stem of the melon grafted onto the squash rootstock ( Figure 6). The results indicated the relative expression levels of six CmGH9Bs, namely CmGH9B2, CmGH9B6, CmGH9B7, CmGH9B14, CmGH9B16, and CmGH9B17, that were significantly upregulated; and the relative expression levels of two CmGH9Bs, namely CmGH9B4 and CmGH9B8, that were significantly downregulated. There were no significant differences for the other 10 CmGH9Bs (CmGH9B1, CmGH9B3, CmGH9B5, CmGH9B9, CmGH9B10, CmGH9B11, CmGH9B12, CmGH9B13, CmGH9B15, and CmGH9B18) under exogenous NAA treatment. Notably, the relative expression level of CmGH9B14, a function analogous to AtGH9B3 ( Figure 1C), was significantly increased with the exogenous NAA application. Nevertheless, further research and evidence are necessary to ascertain whether NAA treatment can enhance the graft compatibility of melon scion and squash rootstock and promote its healing.

Effects of Exogenous Hormones and Far-Red Light Treatment on CmGH9B14 Expression during the Grafting Healing Process
To further verify the expression pattern of CmGH9B14 during the graft healing process, we examined the relative expression of CmGH9B14 with the NAA, SA, and far-red light treatment. In Figure 7, the relative expression levels of CmGH9B14 during the graft healing process of melon scion grafted onto the squash rootstock were significantly higher than that of the own-rooted melon seedlings except for the 8 and 9 DAG. At the 1 DAG, the relative expression level of CmGH9B14 was the highest. From 2 DAG to 9 DAG, the relative expression levels of CmGH9B14 increased first and then decreased. Moreover, it maximized at 4 DAG and was significantly higher than the other days. It inferred CmGH9B14 was very likely to be involved in regulating the grafting healing process of melon scion grafted onto the squash rootstock.

Effects of Exogenous Hormones and Far-Red Light Treatment on CmGH9B14 Expression during the Grafting Healing Process
To further verify the expression pattern of CmGH9B14 during the graft healing process, we examined the relative expression of CmGH9B14 with the NAA, SA, and far-red light treatment. In Figure 7, the relative expression levels of CmGH9B14 during the graft healing process of melon scion grafted onto the squash rootstock were significantly higher than that of the own-rooted melon seedlings except for the 8 and 9 DAG. At the 1 DAG, the relative expression level of CmGH9B14 was the highest. From 2 DAG to 9 DAG, the relative expression levels of CmGH9B14 increased first and then decreased. Moreover, it maximized at 4 DAG and was significantly higher than the other days. It inferred CmGH9B14 was very likely to be involved in regulating the grafting healing process of melon scion grafted onto the squash rootstock.

Effects of Exogenous Hormones and Far-Red Light Treatment on CmGH9B14 Expression during the Grafting Healing Process
To further verify the expression pattern of CmGH9B14 during the graft healing process, we examined the relative expression of CmGH9B14 with the NAA, SA, and far-red light treatment. In Figure 7, the relative expression levels of CmGH9B14 during the graft healing process of melon scion grafted onto the squash rootstock were significantly higher than that of the own-rooted melon seedlings except for the 8 and 9 DAG. At the 1 DAG, the relative expression level of CmGH9B14 was the highest. From 2 DAG to 9 DAG, the relative expression levels of CmGH9B14 increased first and then decreased. Moreover, it maximized at 4 DAG and was significantly higher than the other days. It inferred CmGH9B14 was very likely to be involved in regulating the grafting healing process of melon scion grafted onto the squash rootstock.  There were different expression patterns of CmGH9B14 in the roots, stems, and leaves using the exogenous NAA and SA to treat the own-rooted seedlings of melon (Figure 8). The results showed that both NAA and SA significantly induced the upregulated expression of CmGH9B14 in the melon roots ( Figure 8A) and the downregulated expression of CmGH9B14 in the melon leaves. Then, there was no difference between them. However, in the melon stems ( Figure 8B), only the NAA treatment significantly increased the relative expression levels of CmGH9B14, and there was no difference between the SA and control. Therefore, we speculated that the exogenous NAA treatment accelerated the graft healing process of melon scion grafted onto the squash rootstock by inducing the upregulated expression of CmGH9B14 in the stems of own-rooted melon seedlings.
There were different expression patterns of CmGH9B14 in the roots, stems, and leaves using the exogenous NAA and SA to treat the own-rooted seedlings of melon (Figure 8). The results showed that both NAA and SA significantly induced the upregulated expression of CmGH9B14 in the melon roots ( Figure 8A) and the downregulated expression of CmGH9B14 in the melon leaves. Then, there was no difference between them. However, in the melon stems ( Figure 8B), only the NAA treatment significantly increased the relative expression levels of CmGH9B14, and there was no difference between the SA and control. Therefore, we speculated that the exogenous NAA treatment accelerated the graft healing process of melon scion grafted onto the squash rootstock by inducing the upregulated expression of CmGH9B14 in the stems of own-rooted melon seedlings. To further determine whether far-red light affected the CmGH9B14 expression in the grafting healing process, we treated the grafted melon seedlings with far-red light at 4 DAG. We measured the relative expression levels of CmGH9B14 in the melon scion stems of grafted melon seedlings during the graft healing process. The results indicated that the relative expression levels of CmGH9B14 at 6DAG, 7DAG, and 9DAG after far-red light treatment were significantly higher than those of the control. Far-red light treatment could induce the upregulated expression of CmGH9B14 and advance the expression peak of CmGH9B14 during grafting healing (Figure 9).  To further determine whether far-red light affected the CmGH9B14 expression in the grafting healing process, we treated the grafted melon seedlings with far-red light at 4 DAG. We measured the relative expression levels of CmGH9B14 in the melon scion stems of grafted melon seedlings during the graft healing process. The results indicated that the relative expression levels of CmGH9B14 at 6 DAG, 7 DAG, and 9 DAG after far-red light treatment were significantly higher than those of the control. Far-red light treatment could induce the upregulated expression of CmGH9B14 and advance the expression peak of CmGH9B14 during grafting healing (Figure 9).
There were different expression patterns of CmGH9B14 in the roots, stems, and leaves using the exogenous NAA and SA to treat the own-rooted seedlings of melon (Figure 8). The results showed that both NAA and SA significantly induced the upregulated expression of CmGH9B14 in the melon roots ( Figure 8A) and the downregulated expression of CmGH9B14 in the melon leaves. Then, there was no difference between them. However, in the melon stems ( Figure 8B), only the NAA treatment significantly increased the relative expression levels of CmGH9B14, and there was no difference between the SA and control. Therefore, we speculated that the exogenous NAA treatment accelerated the graft healing process of melon scion grafted onto the squash rootstock by inducing the upregulated expression of CmGH9B14 in the stems of own-rooted melon seedlings. To further determine whether far-red light affected the CmGH9B14 expression in the grafting healing process, we treated the grafted melon seedlings with far-red light at 4 DAG. We measured the relative expression levels of CmGH9B14 in the melon scion stems of grafted melon seedlings during the graft healing process. The results indicated that the relative expression levels of CmGH9B14 at 6DAG, 7DAG, and 9DAG after far-red light treatment were significantly higher than those of the control. Far-red light treatment could induce the upregulated expression of CmGH9B14 and advance the expression peak of CmGH9B14 during grafting healing (Figure 9).

Discussion
Plants are often affected by adverse environmental conditions throughout their life cycle, such as inadequate nutrient supply due to continuous cropping and invasion of various pathogens in the soil. These external environments can interrupt a series of critical physiological and biochemical processes in plants, resulting in growth arrest and even death. However, graft cultivation can significantly improve a single variety's yield, quality, and tolerance. The results showed that the branch of β-1,4-glucanase promoted the cell wall reconstruction near the grafting interface and promoted grafting. The β-1,4-glucanase was found in prokaryotes and eukaryotes. It can efficiently degrade cellulose, complex insoluble substrates, and plant cell wall polysaccharides [21][22][23][24]. In addition, β-1,4-glucanase was involved in cell wall biosynthesis and modification, cell elongation and differentiation, cytokinesis, organ shedding, and fruit ripening [25][26][27][28]. Moreover, the β-1,4-glucanase was also involved in cell wall disintegration during leaf abscission [29]. In recent years, the GH9B gene family of β-1,4-glucanase has played a vital role in promoting graft healing [30] and has attracted more and more attention from researchers in basic and applied plant sciences. In this study, we conducted bioinformatics studies on the vital gene families in the grafting healing process of grafted seedlings and the effects of exogenous substances on family genes to observe the changes of the GH9B gene in the melon grafting healing process. We can conclude and propose that CmGH9B14 may positively affect the grafting healing process of melon scion grafted onto squash rootstock.

Evolutionary Relationships and Characteristics of CmGH9Bs Family Members
GH9B belongs to the E2 subgroups of the glycosidic hydrolase family 9 (GH9) and has the capacity to secrete a single catalytic domain [31,32]. The GH9B genes were identified in Arabidopsis species for their powerful function in solving plant graft healing problems. Comparing the number of GH9B genes in Arabidopsis plants could better analyze the evolutionary relationship among CmGH9Bs and lay the foundation for graft healing. Currently, the GH9B genes reported in rice and Arabidopsis thaliana were most common in Arabidopsis thaliana, which could promote cell adhesion. However, it is not unknown about the information of GH9B genes in the Cucurbit melon.
In this study, 18 CmGH9B genes were identified based on the genomic data of melon. We also established phylogenetic trees to analyze the relationships among GH9B families in melon and Arabidopsis. The results showed that the CmGH9Bs were divided into three families (Figure 1). According to the classification method of Arabidopsis thaliana, CmGH9B1 in melon belonged to Subtribe I, CmGH9B7 and CmGH9B18 belonged to Subtribe II, and Subtribe III contained 15 CmGH9Bs. Generally, GH9B from the same species is more closely related to other species. However, the GH9B genes of the same subfamily were more closely associated with the genes of other subfamilies, suggesting that these genes possibly come from the same ancestor. CmGH9Bs were unevenly located on the melon chromosomes, with chromosome 3 containing the most CmGH9Bs ( Figure 3). According to phylogenetic tree, it seemed that the diversity in GH9B family members had probably occurred before divergences of monocot and dicot [33]. Based on the expression profile, GH9B genes showed diverse expression patterns suggesting that the mutation in regulatory sites of GH9B genes have affected the function and expression of GH9B family members [34,35]. In this study, CmGH9Bs belonged to 18 different response groups detected in the 2.0 KB promoter region of the melon with the help of the PlantCARE tool, among which hormones responding to CmGH9Bs included auxin, GA, ME-JA, and SA ( Figure 4). Among the four types, ME-JA response elements accounted for the most significant proportion of melon promoters, followed by CmGH9Bs associated with auxin, GA, and SA. Cis-acting elements are essential for gene expression, and their number positively correlates with gene expression levels [36]. It is well-known that auxin is a crucial signaling molecule for most organogenesis and gene expression processes during plant growth and development [37]. Thus, we analyzed the effects of exogenous NAA treatment on 18 CmGH9Bs expression in the scion stem of the melon grafted onto the squash rootstock ( Figure 6). The results also showed that the exogenous NAA treatment significantly increased the relative expression levels of CmGH9B2, CmGH9B6, CmGH9B7, CmGH9B14, CmGH9B16, and CmGH9B17.

CmGH9B14 Expression Profiles during the Graft Healing Process
Some reports have shown that the GH9B3 encoding β-1,4-glucanase played an essential role in the graft healing of plants [17][18][19][20]. Through the evolutionary analysis of CmGH9Bs in melon, we found that CmGH9B14 was the homologous gene of AtGH9B3 ( Figure 1C). In this study, we found that the relative expression levels of CmGH9B14 increased first and then decreased during the graft healing process of melon scion grafted onto the squash rootstock ( Figure 7). Furthermore, the CmGH9B14 expression profiles were similar to AtGH9B3, NbGH9B3, and SlGH9B3 [16,18,19], so we speculated that CmGH9B14 was likely to regulate the grafting healing process of the melon scion grafted onto the squash rootstock. However, the molecular mechanism of CmGH9B14 regulating the graft healing process of melon grafted onto squash needs further investigation. In addition, we also analyzed the CmGH9B14 expression changes under the exogenous hormones (NAA and SA) and far-red light treatment during the graft healing process (Figures 8 and 9). The results indicated that the exogenous NAA and far-red light treatment could significantly induce the upregulated expression of CmGH9B14. The results also provided a technical possibility for regulating the expression of CmGH9B14 and then controlling the graft healing of melon grafted onto squash.
In summary, we identified 18 CmGH9B genes, respectively, based on the comprehensive analysis of melon genome data. Genetic information such as chromosomal location and exon-intron structure, conserved domains, and duplicated genes were provided, and the expression profiles of CmGH9Bs were explicitly examined. The CmGH9B14 identified in this study can be used as a candidate gene to promote graft healing, but its specific functions need to be further verified by experiments. Screening candidate genes for graft healing can lay the foundation for improving the quality of grafted seedlings and provide valuable information for better production of grafted seedlings.

Identification and Sequence Analysis of CmGH9Bs Family in Melon
The melon CmGH9Bs were identified using a previously described method but with minor changes. The melon amino acid, genome, CDS sequence assembly, and corresponding annotation were downloaded from the Cucurbitaceae genome database on 15 December 2022 (CuGenDB, http://cucurbitgenomics.org/). The proteins of CmGH9Bs were verified using the Pfam and NCBI databases. Proteins obtained by the domain database screening confirmation were considered CmGH9Bs family members. The corresponding CDS and gene sequences were extracted according to their protein identifications. The MEME program (http://meme-suite.org/) [38] identified conserved motifs of the CmGH9Bs family proteins with the following parameters [39]. The structures of CmGH9Bs were displayed by the TBtools (https://github.com/CJ-Chen/TBtools) software. The chromosomal locations of CmGH9Bs were mapped onto the melon linkage map with an online tool, according to their TIGR numbers. Their molecular weight (Mw), isoelectric point (PI), the number of amino acids, instability index, and other information were subsequently obtained from ExPASy Website (http://www.expasy.org/). Use Euk-mPLoc2.0 (http://www.csbio.sjtu.edu.cn/bionif/euk-multi-2/Plant-mPLoc) to predict the subcellular localization information.

Phylogenetic Analysis
Multiple alignments were performed with the full-length amino acid sequences of CmGH9Bs proteins constructed by phylogenetic trees, using MEGA 7.0 (https://www. megasoftware.net/). Then, the GH9B protein sequence of Arabidopsis thaliana was obtained from the NCBI database, and the phylogenetic trees of melon and Arabidopsis thaliana were constructed similarly.

Chromosomal Mapping and Exon-Intron Distribution
Based on the initial position information of CmGH9Bs in CuGenDB, the chromosomal location images of CmGH9B were drawn by TBtools [37]. The exon-intron distribution of CmGH9Bs was visualized with the help of TBtools (https://github.com/CJ-Chen/TBtools) software.

Cis-Element Analysis of CmGH9Bs
Promoters of the CmGH9Bs family genes were downloaded from the Ensembl Plants database (https://plants.ensembl.org/index.html). The online tools PlantCARE database (https://sogo.dna.affrc.go.jp/) and TBtools software were used to analyze the cis-regulatory elements of the CmGH9B family gene promoters [40].

Plant Growth Conditions and Treatments
The present study involved the application of the splicing graft method to graft the melon (HuaBao, Cucumis melo var. Makuwa Makino) onto the squash (ShengZhen No.1, C. moschata). This experiment was carried out during the scion's first-true-leaf fully expanded stage and the rootstock's cotyledon development stage, utilizing the one-cotyledon method described by Davis et al. [41]. When the scion was just exposed to the heart, we sprayed NAA solution (40 mg·L −1 ) and SA solution (100 mg·L −1 ) 4 times as treatment and sprayed distilled water as control. Grafted seedlings were transplanted in the nutritional bowl (12 cm × 12 cm) and moved into the healing chamber for grafted seedlings' cultivation at Shenyang Agriculture University. At 4 DAG, the grafted seedlings were treated with far-red light for 110 min. The other management methods of grafted seedlings for the control, NAA, SA, and far-red light treatment were consistent [42].

Expression Analysis of CmGH9Bs
The grafting seedlings were divided into melon scion stem and squash rootstock stem. After sampling, the tissues were separated and immediately frozen with liquid nitrogen. After grinding the samples thoroughly in liquid nitrogen, the RNA of the samples was extracted by using an Ultrapure RNA Kit (Kangwei Century, Taizhou, China). An RNA reverse-transcription test was conducted, and the reaction system was as follows: 5x Prime Script RT Master Mix 5 µL, RNA 1000 ng, and ddH 2 O refill to 20 µL. The reaction procedure was 37 • C for 15 min, 85 • C for 5 s, and 4 • C for cooling. After the reaction, the obtained cDNA was diluted four times and then stored at −20 • C for subsequent qRT-PCR and gene cloning tests.
The transcriptional levels of different CmGH9Bs in three tissues of melon were measured using qRT-PCR, and the transcription levels of grafted seedlings with the exogenous hormones (NAA and SA) and far-red light treatment were also determined. The qRT-PCR primers of CmGH9Bs were designed using Primer 5 (http://frodo.wi.mit.edu/).The internal reference gene uses β-actin. The qRT-PCR experiments were carried out via 2 × SYBR Green Pro Taq HS Premix (TaKaRa, Beijing, China). The gene expression data were calculated by the 2 −∆∆ CT method [43], the qRT-PCR experiments were carried out based on three biological replicates, three technical replicates were carried out for each biological replicate, and pictures were drawn using Prism 8.3.0. software.

Statistical Analysis
All experimental data, including those for correlation analyses, were processed using DPS statistical software V26.0 and expressed as the mean of three biological replicates of RNA sequence results or the mean ± standard deviation (SD) of three biological replicates. Differences between melon and squash samples were assessed at a significance level of 0.05 by one-way ANOVA tests.

Conclusions
In this study, the CmGH9Bs of melon were characterized at the genomic level. By studying the gene structure and motif composition of 18 CmGH9B genes, their evolutionary relationship with Arabidopsis GH9B genes, and their expression in different tissues of grafted seedlings, candidate genes that may promote graft healing were identified. In addition, the effects of exogenous hormones and far-red light treatment on CmGH9Bs expression profiles were preliminarily verified. Based on our analysis, we hypothesized that CmGH9B14 was likely to be a vital regulator of the graft healing process in melon scion grafted onto the squash rootstock. Although the functions of these candidate genes need to be confirmed, this study laid a solid foundation for promoting grafting healing and producing high-quality grafted seedlings.